FXR was previously identified as a nuclear receptor activated by molecules possessing farnesyl backbones. We have continued work on this project to characterize the physiological activities controlled by farnesoids and FXR by isolating farnesol-regulated genes. Dissociated mouse liver cells were seeded in culture dishes and incubated either with 50 uM farnesol in 0.5% methanol or with methanol alone for 3, 8, or 17 hours. RNAs were isolated from treated cells and used to program cDNA synthesis. The cDNAs from each sample were amplified using the polymerase chain reaction and specific oligonucleotide primer sets. Three distinct farnesol-regulated gene fragments were identified from more than one hundred primer set combinations. The gene fragments are presently being subjected to DNA sequencing. As a second aspect of this project, an exon corresponding to the second half of the FXR DNA-binding domain was isolated from a mouse genomic DNA library. This FXR-encoding exon was disrupted by substituting a phosphoglycerate kinase promoter-driven neomycin gene element. The herpes virus thymidine kinase gene was introduced downstream of a 1.8 kilobase pair FXR genomic fragment to create a targeting vector to be used for selecting FXR-disrupted embryonal stem cells. Blastocysts will be injected with these cells to create animals with FXR-disputed genes.